Al-Azhar Assiut Medical Journal

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 17  |  Issue : 1  |  Page : 9--13

Peripheral blood mononuclear cells hepatitis C virus RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients from the Damietta Governorate


Alaa E Hashim1, Samy Zaky2, Naglaa Azab3, Fathiya El-Raey1, Mahmoud A Halim4,  
1 Tropical Medicine Department, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
2 Tropical Medicine Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt
3 Medical Biochemistry Department, Faculty of Medicine, Banha University, Benha, Egypt
4 Tropical Medicine Department, Damanhour Fever Hospital, Ministry of Health, AL-Behera, Egypt

Correspondence Address:
Mahmoud A Halim
Damanhur Fever Hospital, Ministry of Health, AL-Behera, 22551
Egypt

Abstract

Background Eradication of hepatitis C virus (HCV) infection is the goal of direct-acting antivirals with a high rate of sustained virological response (SVR). Currently, SVR is determined by negative serum HCV RNA by real-time (RT)-PCR that may not give any information about intracellular HCV replication. Aim To study peripheral blood mononuclear cells (PBMCs) HCV RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients. Patients and methods In all, 150 patients with chronic HCV, classified into 100 easy-to-treat patients (group I) and 50 difficult-to-treat patients (group II), who achieved SVR to sofosbuvir plus daclatasvir with or without ribavirin were enrolled in the study. HCV RNA was evaluated in both sera and isolated PBMCs using the polymerase chain at 3 and 12 months from the end of treatment (EOT). Results As regards HCV RNA in peripheral blood mononuclear cells (PBMCs) we found that no case was positive in the easy to treat (group I). In difficult to treat (group II), six (4%) patients were positive at 3 months and 24 (16%) patients at 12 months after EOT. However, there is no virological, clinical, or biochemical relapse noted during the follow-up period among positive cases. All positive cases were cirrhotic, with significantly lower platelets count and albumen level but higher bilirubin than group I. Conclusion All easy-to-treat groups were HCV RNA in PBMCs negative at EOT and during the follow-up period (1 year). Follow-up of cirrhotic patients with positive HCV RNA in PBMCs showed no clinical, biochemical, or virological relapse.



How to cite this article:
Hashim AE, Zaky S, Azab N, El-Raey F, Halim MA. Peripheral blood mononuclear cells hepatitis C virus RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients from the Damietta Governorate.Al-Azhar Assiut Med J 2019;17:9-13


How to cite this URL:
Hashim AE, Zaky S, Azab N, El-Raey F, Halim MA. Peripheral blood mononuclear cells hepatitis C virus RNA as a predictor for the response to daclatasvir-containing oral antiviral regimen in chronic hepatitis C patients from the Damietta Governorate. Al-Azhar Assiut Med J [serial online] 2019 [cited 2020 Mar 31 ];17:9-13
Available from: http://www.azmj.eg.net/text.asp?2019/17/1/9/266740


Full Text



 Introduction



HCV has the ability to infect not only hepatocytes, but also peripheral blood mononuclear cells (PBMCs) [1].

The presence of HCV RNA in hepatocytes or PBMCs of patients with negative for serum HCV RNA is defined as occult HCV infection, which may be cryptogenic or secondary. Occult HCV infection is best identified by the detection of intracellular viral genomic RNA in hepatocytes or PBMCs [2].

PBMCs are considered reservoirs of HCV RNA. Detection of replicative forms of HCV RNA in PBMCs has been demonstrated in some cases [3].

The goal of the HCV therapy is to eradicate the infection and prevent the progression of the disease. Eradication of HCV infection is achieved by direct-acting antivirals (DAAs) with a high rate of sustained virological response (SVR).

Most HCV relapses that occur following the currently used DAA therapy occur within weeks to years after the end of treatment (EOT) [4]

Detection of HCV RNA in PBMCs at end of treatment (EOT) predicts HCV relapse within 12 weeks after EOT with interferon plus ribavirin regimen [5],[6],[7]

Testing for HCV RNA by both SRT-PCR and PBMCs PCR is recommended and expected to give more information than serum HCV RNA only [8].

 Aim



To study the impact of using PBMCs HCV RNA as a predictor for the response to treatment by daclatasvir/sofosbuvir regimen 1 year after the EOT in patients with chronic hepatitis C in the Damietta Governorate.

Patients

This study was conducted on 150 patients with chronic hepatitis C-related liver disease. All patients were eligible for treatment according to the protocol of treatment of chronic HCV, established by the Egyptian National Committee for Control of Viral Hepatitis and launched in December 2016 [9]. Patients had received combined, oral, generic antiviral therapy, in the form of sofosbuvir (400 mg, tablets), one tablet, plus daclatasvir (60 mg, tablets), one tablet, with or without generic ribavirin (200 mg, capsules), daily, for 12 weeks. All patients received treatment in the Unit of Viral Hepatitis, Fever Hospital, Damietta, Egypt.

Inclusion criteria

Patients achieved serum HCV RNA clearance and successful SVR responses.

Patients classification

The patients were categorized into two groups:Group I (easy to treat):This group included 100 patients, who are treatment naive with the level of serum total bilirubin being up to 1.2 mg/dl, serum albumin more than or equal to 3.5 g/dl, international normalisation ratio (INR) up to 1.2, and platelets count more than or equal to 150×103/mm3.Group II (difficult to treat): including 50 patients, being with either of pegylated interferon (peg-IN) treatment experienced or level of total serum bilirubin more than 1.2 mg/dl or serum albumin less than 3.5 g/dl, INR more than 1.2, and platelets count of less than 150×103/mm3

Evaluation of treatment outcome

Using PCR assay, quantitative HCV viral load testing was evaluated in serum samples at 12 week after EOT to detect SVR and at 48 weeks following completion of therapy to detect achievement of virological response after 1 year of treatment.PBMC HCV RNA was tested in plasma samples at 12 and 48 week after EOT to detect virological response to treatment.

Testing for HCV RNA

Quantitative HCV RNA was detected by real-time PCR assay HCV Rotor-Gene Real-Time (RT-qPCR) kit with a lower limit of quantification of 15 IU/ml.PBMC HCV RNA: the blood sample was collected in a sterile vacutainer containing EDTA as an anticoagulant. Immediately after collection, PBMCs were separated by Ficoll density gradient centrifugation and isolated using Ficoll-hypaque separation medium (GE Healthcare Life Sciences, Chicago, Illinois, USA). HCV RNA extraction from PBMCs was done by reverse transcription RNA. Detection of HCV RNA was by real-time PCR assay. HCV RNA was extracted from PBMC samples using PROBA-RNA/DNA Mini Kit (100T) DNA-Technology, Research and Production (Moscow, Russia) according to manufacturer’s instructions [10].

Statistical analysis

The data were collected, coded, and analyzed with the program statistical package for the social sciences (SPSS, IBM®, Chicago, Illinois, USA), under Windows version 22 as follows:Description of the quantitative variables in the form of mean±SD.Description of the qualitative variables in the form of frequency and percentage.Student’s t-test: for two independent samples is used to compare quantitative variables.One-way analysis of variance test: for comparison between multiple groups with quantitative continuous variables.χ2-Test: used to compare a qualitative variable between two or more independent groups.

For interpretation of results, significance level (P) value was expressed as follows:P≤0.05=significant.P>0.05=nonsignificant.

 Results



Baseline characteristics

A total of 150 Egyptian patients were included in this study with a mean age of 52.75±9.87 years.There were 72 (48%) men and 78 (52%) women.Mean of BMI was 30.35±5.96 kg/m2.There were 18 (18%) controlled diabetics in group I and seven (14.0%) in group II.Variable baseline biochemical and virological characteristics of the enrolled 150 patients were analyzed ([Table 1]){Table 1}Assessment of virological response at 12 week EOT Samples of the patient’s serum were tested for HCV RNA by real-time PCR assay. HCV RNA was not detected in the serum of all patients. Thus, all 150 patients (100%) achieved a successful SVR response.PBMC: HCV RNA was not detected in plasma samples of all easy-to-treat patients (group I), but it was detected in the samples of six (4%) patients among difficult-to-treat patients (group II).There was statistically significant difference between the studied groups ([Table 2]).{Table 2}Assessment of virological response at 48 week after EOT:All enrolled 100 easy-to-treat patients continue to maintain successful response to treatment with undetectable HCV RNA in the serum.All 100 easy to treat patients continue to maintain negative PBMC HCV RNA. But another 18 (12%) patients among the difficult to treat group showed positive PBMC HCV RNA.There was statistically significant difference between the studied groups ([Table 3]).{Table 3}Conclusively, we found that all positive cases were old age, cirrhotic patients with high HCV viremia and previous HCV treatment (PEG INF) experience (nonresponder/failure) ([Table 4] and [Table 5]).{Table 4}{Table 5}

 Discussion



New DAAs are highly effective and safe in the eradication of chronic HCV infection. The goal of DAAS is achievement of SVR. SVR rates for DAA treatment are more than 90% in published trials [11]. However, relapse following the EOT by the antiviral therapy may occur [12].

HCV has ability to infect multiple cell types other than hepatocytes. HCV RNA can be detected in PBMCs of infected individuals [13]. Patients cured after antiviral treatment with persistent viral response may have occult HCV infection in the liver or PBMCs. So, the presence of HCV in PBMCs may be one of the causes of relapse after antiviral therapy. Perhaps clearance of HCV RNA from PBMCs could be a predictor for persistent response to antiviral therapy and a reference to formulate the duration of antiviral therapy in chronic hepatitis C [12].

To study the PBMC HCV RNA as a predictor of response to daclatasvir containing oral antiviral regimen in chronic hepatitis C patients from the Damietta Governorate. A total of 150 patients achieved serum HCV RNA clearance and successful SVR responses were enrolled in this study.

We found that PBMCs HCV RNA was negative in all easy to treat (group I) patients, at week 12 after EOT, and at the end of study, while six (4%) patients at week 12 after EOT and 24 (16%) patients at 12 months after EOT among the difficult to treat (group II) had positive PBMCs HCV RNA. Our result is nearly comparable with Gallegos-Orozco et al. [14], who reported that HCV RNA was positive in PBMCs of 20% cases with clinical SVR to INF and also with Abu Khadr Nouh et al. [15] who detected HCV RNA in PBMCs in 25% of patients treated with a sofosbuvir-daclatasvir (SOF-DACLA) regimen. Abd Alla et al. [16] detected HCV RNA in PBMCs in 18.07% of patients treated with different combinations of different DAAs.

Analysis of the characteristic features of patients who had positive PBMCs HCV RNA concluded that we found that all positive cases were old age, cirrhotic patients with high HCV viral load, and previous HCV treatment (PEG INF) experience (nonresponder/failure). This may be explained by the impact of special circumstances, for example, immunosuppression [14].

Marukian et al. [17] in an in-vitro culture study showed that ∼0.1% of the input viral RNA remained associated with PBMC cultures after the input virus was extensively washed away. Also, the level of HCV RNA did not increase in cultures of PBMCs at any time, and that the low level of HCV RNA detected in PBMCs was not affected by 2′CMA, an inhibitor of HCV RNA-dependent RNA polymerase. This raised a question whether HCV indeed infects and replicates in PBMCs or just associates with PBMCs without undergoing replication.We followed up a total of 150 cases for up to 1 year after completion of course of HCV treatment, and we found no clinical, biochemical, or virological relapse and all cases continued to maintain their successful response to treatment with clearance of HCV RNA from their serum. This may be attributed to that viral RNA may be bound or taken up by cells without undergoing a complete infectious cycle and suggested that detection of HCV RNA associated with blood cells does not prove that the virus replicates in these cells [17].

 Conclusion



All easy-to-treat groups had negative HCV RNA in PBMCs at 12 weeks after EOT and during follow-up at 48 weeks.

Follow-up of cirrhotic patients with positive HCV RNA in PBMCs showed nonsignificant clinical, biochemical, or virological relapse.

Acknowledgements

The authors acknowledge the National Committee for the Control of Viral Hepatitis, Egypt for approving this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Lamelin JP, Zoulim F, Trepo C. Lymphotropism of hepatitis B and C viruses: an update and a newcomer. Int J Clin Lab Res 1995; 25:1–6.
2Carreno V, Pardo M, Lopez-Alcorocho JM, Rodriguez-Inigo E, Bartolome J, Casstillo I. Detectionof hepatitis C virus (HCV) RNA in the liver of healthy, anti- HCV antibody-positive, serum HCV RNA-negative patients with normal alanine aminotransferase levels. J Infect Dis 2006; 194:53–60.
3Pawe1czyk A, Kubisa N, Jabłońska J, Bukowska-Ośko I, Caraballo Cortes K, Fic M et al. Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3+, CD14+ and CD19+. Virol J 2013; 10:346.
4Chou R, Hartung D, Rahman B, Wasson N, Cottrell EB. Comparative efectiveness of antiviral treatment for hepatitis C virus infection in adults: a systematic review. Ann Intern Med 2013; 158:114–123.
5El-Awady MK, Abdel Rahman MM, Ismail SM, Amr KS, Omran M, Mohammed SA. Prediction of relapse after interferon therapy inhepatitis C virus-infected patients by the use of triple assay. J Gastroenterol Hepatol 2003; 18:68–73.
6Zaman N, Asad MJ, Raza A, Raja GK, Akhter S, Mahmood M, Mahmooda RT. Presence of HCV RNA in peripheral blood mononuclear cells may predict patients’ response to interferon and ribavirin therapy. Ann Saudi Med 2013; 34:401–406.
7Hanno AFF, Mohiedeen KM, Alshayeb AF, Deghedy A. HCV RNA in peripheral blood mononuclear cells (PBMCs) as a predictor of the response to antiviral therapy in chronic hepatitis C. Alexandria J Med 2014; 50:317–322.
8Abd Alla MD, El Awady MK. Hepatitis C virus RNA strands detection in peripheral blood mononuclear cells legitimizes virus eradication in negative serum PCR naive and post-treatment patients. J Clin Transl Hepatol 2017; 5:1–8.
9El-Akel W, El-Sayed MH, El Kassas M, El-Serafy M, Khairy M, Elsaeed K et al. National treatment programme of hepatitis C in Egypt: hepatitis C virus model of care. Journal of viral hepatitis 2017, 262–267.
10Jaatinen T, Laine J. Isolation of mononuclear cells from human cord blood by Fico-Paque density gradient. Curr Protoc Stem Cell Biol 2007; 1:2A1.1–2A1.4.
11Kohli A, Shaffer A, Sherman A, Kottilil S. Treatment of hepatitis C: a systematic review. JAMA 2014; 312:631–640.
12Pan YF, Qin T, Jiang HQ, Wu SH, Gu JS. Detection of hepatitis C virus in serum and peripheral blood mononuclear cells by two reverse transcription polymerase chain reactions. Chin Med J (Engl) 2007; 120:431–433.
13McKeating JA, Zhang LQ, Logvinoff C, Flint M, Zhang J, Yu J et al. Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81-dependent manner. J Virol 2004; 78:8496–8505.
14Gallegos-Orozco JF, Rakela J, Rosati M, Vargas HE, Balan V. Persistence of hepatitis C virus in peripheral blood mononuclear cells of sustained virological responders to pegylated interferon and ribavirin therapy. Dig Dis Sci 2008; 53:2564–2568.
15Abu Khadr Nouh HH, Hanafi NF, Sara L, Asser SA, Hussain YA. Secondary occult hepatitis C virus infection (HCV) in chronic HCV patients after treatment with sofosbuvir and daclatasvir. Int J Curr Microbiol App Sci 2018; 7:1357–1365.
16Abd Alla MD, Elibiary SA, Elshaboury RH, Wu GY, Dawood RM, El Awady MK. HCV Therapy follow-up fractionation (CTF2) by intra-PBMC nested RNA PCR recognizes early virologic response and relapse. J Clin Transl Hepatol 2018; 6:1–8.
17Marukian S, Jones CT, Andrus L, Evans MJ, Ritola KD, Charles ED et al. Cell culture-produced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 2008; 48:1843–1850.